Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells

Abstract

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

“…Despite the potential of hPSC-EVs, we observed that the number of publications in this area is still low, and most of the existing publications date from the last five years (Figure 1A , Table 1 ). Some studies evaluate EVs that were isolated during the differentiation process or from cells that differentiated from PSCs, such as hiPSC-derived keratinocytes[ 38 ]; hPSC-derived cardiac progenitors or cardiomyocytes[ 39 - 41 ]; hPSC-derived MSCs[ 42 - 44 ]; hiPSC-derived neurons[ 45 - 47 ]; and hESC-derived chondroprogenitor cells[ 48 ]. However, our review explores studies that isolated EVs from undifferentiated hPSCs.…”

“…Even though many articles described the effects of hPSC-EVs, few made deeper characterizations of, for example, the protein and miRNA content of these EVs. Some performed proteomic analysis to help explain some of the effects[ 59 ] or as a control (time 0) to study the differentiation process[ 39 , 46 ]. In one interesting approach using high-density lectin microarray, Saito et al [ 57 ] demonstrated that rBC2LCN, a specific lectin for hPSCs, bound to hiPSC-derived EVs but not to adipose-derived stem cell-, hemodiafiltration-, or chondrocyte-derived EVs, which suggests a particular glycan-signature for hiPSC-EVs, resembling the glycome signature of the cell surface.…”